Integration of BMP, RTK/MAPK and Wnt/GSK3 signals through Smad1 phosphorylations.

Understanding how cells integrate distinct signaling pathways to achieve simple cell differentiation outcomes is one of the major challenges in developmental biology. The undifferentiated Xenopus ectodermal cell constitutes a powerful system to study signal integration because several signaling pathways converge to regulate the decision to acquire either neural or epidermal fates. In the embryo, the inhibition of BMP signaling by antagonists such as Chordin and Noggin results in neuralization of the ectoderm. However, neural differentiation can also occur by dissociating the ectodermal cells in the absence of an inducing signal. This "default" neural induction is caused by sustained activation of the Ras/MAPK pathway, triggering the inhibition of the BMP transducer Smad1, through phosphorylation at conserved sites in its linker region. Upon further analysis of the linker region, I identified that Smad1 is also phosphorylated by GSK3, a kinase involved in the Wnt pathway. This is of particular interest since inhibition of the Wnt pathway is required for neurogenesis. MAPK and GSK3 phosphorylations are required for Smad1 polyubiquitinylation and degradation by the proteasomal machinery. Importantly, Wnt signaling decreased GSK3-dependent phosphorylation of Smad1. Therefore, the RTK/MAPK and Wnt/GSK3 signaling pathways control the duration of the BMP signaling through Smad1 phosphorylation and degradation. This signal integration mechanism explains why neural differentiation takes place when Smad1 signaling is at its lowest (low BMP, high MAPK and low Wnt). Following these findings, I analyzed linker-phosphorylated Smad1 in a variety of cultured cell lines. Unexpectedly, I found that during cell division of human Embryonic Stem Cells (hESC) and other mammalian cells such as Cos7, there is an asymmetric distribution of proteins targeted for degradation like phosphorylated Smad1, phosphorylated beta-Catenin and total polyubiquitinylated proteins. Similar asymmetries were observed in Drosophila embryos. These findings suggest that we have identified a new cellular mechanism in which proteins destined for degradation segregate asymmetrically during mitosis in order to maintain a daughter clear of undegraded proteins. The work presented in this dissertation suggests that the use of Smad1 as a platform to integrate multiple signaling pathways provides a molecular explanation for self-regulating properties of fields of cells undergoing differentiation during early development.